A dynamic cycle of addition and removal of O-GlcNAc in the nucleus and cytoplasm mediates a final step in the hexosamine signaling pathway. The targets of this modification are nuclear pore complexes, transcription complexes, proteasomes and signaling kinases. Based on the targets modified by O-GlcNAc, we proposed that the enzymes of O-GlcNAc metabolism modulate nuclear transport, transcription, cell growth, and apoptosis in response to nutrient availability. Examining the structure, targeting, and regulation of the enzymes of O-GlcNAc metabolism is our principal focus. O-GlcNAc is transferred to proteins from UDP-GlcNAc, a sugar nucleotide whose levels are regulated by the hexosamine biosynthetic pathway (HBP) acting as a cellular sensor of nutrient availability. By integrating these signals, the HBP regulates expression of a number of gene products that include leptin. In skeletal muscle, flux through the HBP correlates with the degree of insulin resistance. The HBP is also linked to pathways regulating cell proliferation and apoptosis; fibroblasts that cannot acetylate UDP-GlcNAc exhibit defects in proliferation, adhesiveness and resistance to apoptotic stimuli. Thus, by generating UDP-GlcNAc, the HBP may be viewed as a nutrient-sensing signaling pathway. We seek to determine how O-GlcNAc participates in this signaling cascade. We are testing the hypothesis that differentially targeted isoforms of the enzymes of O-GlcNAc metabolism mediate this glycan-dependent signaling pathway. By responding to nutrient levels, this pathway modulates gene expression, cell growth and programmed cell death. We expressed fully functional OGT and O-GlcNAcase isoforms in E. coli. We recently solved the structure of the superhelical TPR (tetratricopeptide repeat) domain of OGT that mediates the recognition of target proteins and showed that exhibits structural similarities to importin alpha. Consistent with a role as a signaling molecule, we showed that OGT modifies glycogen synthase kinase-3 and casein kinase, two enzymes regulating glycogen synthesis. Much of the impact of O-GlcNAc cycling occurs through changes in transcription. Importantly, both histone deacetylases (HDAC) and OGT are recruited to Sin3a transcription-repression complexes. The C-terminus of one isoform of the O-GlcNAcase has been shown to be a histone acetyltransferase (HAT). Therefore, O-GlcNAc appears to be a dynamic participant in histone remodeling complexes. We are currently testing this hypothesis by interfering with O-GlcNAc cycling and examining the subsequent impact on chromatin using CHIP-on-Chip, high throughput sequencing and expression array technologies. We have also focused on the catalytic functions of the enzymes of O-GlcNAc cycling. We demonstrated that both isoforms of O-GlcNAcase are active enzymes modulating cellular O-GlcNAc levels. Mutational analysis of OGT and O-GlcNAcase allowed us to define catalytic domains. We showed that OGT isoforms are targeted to both nucleus and mitochondria. The differential localization of mitochondrial and nuclear isoforms of OGT argues that they perform unique intracellular functions in apoptosis, mitochondrial movement and transcriptional repression respectively. O-GlcNAcase isoforms are also differentially targeted in cells; one isoform is nuclear while another accumulates at cellular sites of lipid storage. Small molecule inhibitors and substrates for the enzymes of O-GlcNAc metabolism are under development using both synthetic and natural product approaches. We have proposed that this intracellular glycan modification of Ser/Thr participates in diverse signaling pathways in a manner analogous to protein phosphorylation. We also have taken Chemical Biology approaches to examine O-GlcNAc cycling. We first developed a chemical method for detecting O-GlcNAc addition. This method is being used for high throughput screening to identify inhibitors of O-GlcNAc cycling. We have also developed a number of O-GlcNAcase-specific fluorogenic substates and inhibitors that will facilitate dissection of the hexosamine signaling pathway implicated in Type-2 diabetes, obesity and neurodegeneration. These reagents will facilitate the dissection of the nutrient-sensing hexosamine signaling pathway. The enzymes of O-GlcNAc cycling may play a key role in the changes in signaling and epigenetic landscape associated with metabolic disease.